Acridinium esters (AE) provide for an extremely sensitive method of detection and are useful chemiluminescent signal molecules that have been used extensively as labels in immunoassays and nucleic acid assays. U.S. Pat. Nos. 4,745,181; 4,918,192; 5,110,932 first described hydrolytically stable Polysubstituted Aryl Acridinium Esters (PAAE) which are useful for analytical measurements and became the first chemiluminescent acridinium compounds that enabled ligand binding assays to meet the stringent conditions required for commercialization owing to their remarkable stability. Subsequently, U.S. Pat. Nos. 5,241,070; 5,538,901; and 5,663,074 described nucleophilic PAAE useful for the direct labeling of diverse organic molecules which lack nucleophilic functional groups. The utility of PAAE was further enhanced with the advent of Functionalized Hydrophilic PAAE (U.S. Pat. No. 5,656,426) which increased the quantum yield of PAAE and enhanced the performance of PAAE-labeled binding partners in terms of the observed signal to noise ratios and the sensitivities of various binding assays. This was primarily due to the introduction of hydrophilic group at the acridinium nucleus which increased the aqueous solubility of the compound and also unexpectedly increased the quantum yield of light production. Additionally, introduction of ionizable groups at the phenoxy moiety produced another sub-class of hydrophilic PAAE (U.S. Pat. Nos. 5,227,489; 5,449,556; and 5,595,875) which could be encapsulated in large numbers within biomolecule-functionalized, liposomes with extremely low leakage over prolonged storage. The last application further enhanced the utility of PAAE.
M. Kawaguichi, et al. (Bioluminescence and Chemiluminescence, Proceedings of 9th International Symposium 1996, Ed. Hastings, Kricka and Stanley, John Wiley & Sons, 1997, pp. 480–484) have described stabilized phenyl acridinium esters for chemiluminescent immunoassays. AE derivatives with additional methyl substitutions at C-1, which are optional at C-3 of the acridinium nucleus with matching mono- or di-methyl substitutions at the ortho-positions of the phenoxy moiety, were shown to have excellent stability in aqueous solution.
EP 0324,202 A1 and subsequently EP 0609,885 A1 both describe acridinium esters with functional groups substituted at the nitrogen atom of the acridinium nucleus. The latter application further describes alternate substituents such as the biphenyl or naphthyl moieties as possible replacements for the phenyl group. These types of acridinium compounds are reported to have emission maxima at 420 nm.
Mattingly, et al. (U.S. Pat. Nos. 5,468,646 and 5,543,524) describe chemiluminescent acridinium salts, their methods of preparation, their antibody conjugates, and their applications in immunoassays. These acridinium salts belong to another class of compounds termed acridinium sulfonylamides (or N-sulfonylacridinium carboxamides). The acridinium sulfonylamides (AS) have aqueous stabilities which are comparable with PAAE. No emission maxima were reported for the AS described therein. However, since the same acridone species should be generated from both classes of these compounds during their reaction with alkaline peroxide, the emission maxima for the acridinium sulfonylamides is expected to be in the blue region.
Mattingly, et al. further describe and claim the analogous chemiluminescent phenanthridinium salts, their methods of preparation, their antibody conjugates, and their applications in immunoassays, in U.S. Pat. Nos. 5,545,739; 5,565,570, and 5,669,819. Additionally, in these patents a general structure of acridinium sulfonylamides is described showing possible substitutents of a Markush group at the acridinium nucleus. No particular benefits about the substitutents were stated. None of the AS derivatives depicted by the general structure fits the teachings described by the present invention. Finally, the above patents do not describe any attempt to extend the wavelength of light emission of the acridinium sulfonylamides nor do they outline any strucuture-activity rationale about how this may be achieved.
Conventional acridinium compounds, such as those described in the aforementioned patents and literature, emit light with maxima at about 428 nm upon reaction with hydrogen peroxide in strong alkaline solution. Acridinium compounds which emit light of wavelength maxima >500 nm have also been described in the prior art. U.S. Pat. Nos. 5,395,752; 5,702,887 and 5,879,894 by Law et al. describe novel, long-emission acridinium esters (LEAE), where a fused, benzacridinium system is employed to extend the wavelength of emission of the acridinium ester. In the copending PCT application PCT/IB98/00831, Jiang et al. have further extended the PAAE emission maxima well into the region of 600–700 nm by utilizing the principle of energy transfer. This entailed the covalent coupling of luminophores to acridinium ester. When the chemiluminescent reactions of these conjugates were initiated by treatment with alkaline peroxide, light emission was observed at long wavelengths where the wavelength maxima depended upon the structure of the luminophore.
EP 0 478 626 B1 and U.S. Pat. No. 5,656,207 by Batmanghelich et al. outline a structure for a purported, long-wavelength-emitting acridinium ester, in which an extended conjugation system is drawn by appending a substituted carboxybutadienyl group to the acridinium ester. However, in the Batmanghelich patents, neither the synthesis of this acridinium ester nor its emission properties was described to enable and substantiate the claim of light emission maxima of 500–700 nm, as already pointed out in the U.S. patent application Ser. No. 08/308,772.
Other non-acridinium ester-based, long emitting chemiluminescent compounds related to stable 1,2-dioxetanes have been described by Bronstein et.al. in U.S. Pat. No. 4,931,223. Said patent discloses chemiluminescent 1,2-dioxetanes comprised of enzyme-cleavable, functional groups and light emitting fluorophores with different emission wavelengths. Specific preferred embodiments include a acetoxybenzopyran-substituted stable dioxetane (A), a phosphoryloxy-benzopyran-substituted stable dioxetane (B), and a β-galactosyloxy-benzothiazolyl-benzopyran-substituted stable dioxetane (C). The dioxetane A emits light with a 450 nm wavelength maximum when its acetoxy group is cleaved by an esterase. The dioxetane B emits lights at a 480 nm wavelength maximum when its phosphoryloxy group is cleaved by a phosphatase, while the dioxetane C emits light at 515 nm wavelength maximum upon treatment with the enzyme β-galactosidase. The patent provides an example of a three-channel analysis for the simultaneous detection of HSV, CMV, and HPV in a nucleic acid probe simultaneous assay, using three, narrow band-pass optical filters to sort out the different color emissions from the aforementioned dioxetanes. The levels of HSV, CMV, and HPV present in the sample were correlated by the corresponding image brightness. Because the three light emission maxima are so close together, most of the spread from each emission spectrum had to be cut off by the narrow band pass filters to remove signal from the overlapping regions. This resulted in a very little usable amount of signal for each assay component and greatly limited the assay sensitivity and perhaps accuracy.
Edwards et al. [J. Biolumin. & Chemilumin., 5, 1 (1990)] have reported an another chemiluminescent dioxetane, 3-(2′-spiroadamantane)-4-methoxy-4-(7″-acetoxy)naphth-2′-yl-1,2-dioxetane, which emits green light (maximum of 550 nm) with a bathochromic shift of 90 nm in comparison to its 6″-acetoxy substituted isomer. This is attributed to the different position of the enzymatically cleavable acetoxy substituent that gives rise to an oxide anion substituent responsible for triggering dioxetane decomposition. Similar application of the two isomeric dioxetane compounds for the simultaneous detection of several analytes was suggested in the paper.
In this invention, we describe the design and synthesis of novel acridinium compounds which emit light with wavelength maxima >590 nm upon reaction with hydrogen peroxide. These acridinium compounds contain some key structural features which are critical for observing long-wavelength emission. These results along with our earlier observations described in U.S. Pat. No. 5,395,752 provide sound and experimentally verified rules for the design and synthesis of long-wavelength-emitting acridinium compounds.
For the improved measurement of the NIR chemiluminescent signal we also disclose in the present invention a modified, semi-automatic luminescence analyzer in which the red-insensitive photomultiplier tube has been replaced with a state-of-the-art low-noise, cooled CCD detector utilized in photon counting mode. By comparing the quantitated signals obtained from the original and modified analyzers we demonstrated an improvement in the specific activity of a NIR acridinium compound by about 40-fold. The use of a cooled CCD camera system for imaging chemiluminescent signal in the short wavelength region generated from a 1,2-dioxetane compound, was described by Martin, et.al. in J. Biolumin. Chemilumin. 9 (3), 145, 1994. The applications that were said to have been adapted to this imaging method, included various nucleic acid and immuno blottings, ELISA methods and DNA sequencing systems.